New Chromosomal AmpC β-Lactamase in Enterobacter cloacae

Several members of the Enterobacteriaceae, including Enterobacter spp., are naturally resistant to amoxicillin and cephalosporins. Enterobacter cloacae produces chromosomally encoded β-lactamases, also called cephalosporinases (1), and is a serious nosocomial pathogen, the third most prevalent bacterium isolated in intensive care settings (5, 8). We report here the study of a new chromosomal AmpC β-lactamase produced by E. cloacae FFUL2En isolated from the blood culture of a patient hospitalized in a medicine ward of Hospital de Santa Maria, Lisbon, Portugal. The antibiogram revealed resistance to aminopenicillins, aztreonam, and broad-spectrum cephalosporins, except imipenem, aminoglycosides, and quinolones. By isoelectrofocusing, the sonicate extracts expressed a pI of 8.68, suggesting the presence of a presumed AmpC enzyme.

A total DNA preparation from E. cloacae FFUL2En was used in PCR experiments with two sets of primers, TN5 (5′-CGTTTGTCAGGCACAGTCAAATCCA) and TN4 (5′-TTACTGTAGCGCGTCGAGGATATGG) and the internal primers TN2 (5′-TTCCACTGCGGCTGCCAGT) and TN3 (5′-CGGATGAGGTCACGGATAACGCC), designed in accordance with consensus sequences from the ampC genes described for E. cloacae and available at GenBank. The amplicon with 1,234 bp was cloned into the SmaI site of the pBK-CMV vector (6) with a TOPO TA cloning kit, resulting in the plasmid p2En1. The β-lactam susceptibility pattern of Escherichia coli 2En1, harboring the recombinant plasmid p2En1, displayed cefoxitin, cefuroxime, ceftazidime, and piperacillin plus tazobactam MICs of >256 μg/ml and a cefepime MIC of 0.5 μg/ml. The MICs of cefotaxime and aztreonam were lower than those for the parental strain (Table 1). The E. coli 2En1 transformant showed the same pI as the parental strain (pI 8.68), and the substrate profile of the enzyme EcloFFUL2En was determined with the transformant crude enzymatic extract (7). The V_max values indicate that cephalothin, with a V_max of 3,000.1 μM/min, is hydrolyzed more quickly than cefoxitin (V_max = 3.7 μM/min). Ceftazidime and cefotaxime are not hydrolyzed at detectable levels (V_max = <0.1 μM/min).

TABLE 1.

  MICs of β-lactams for E. cloacae FFUL2En clinical isolate, E. coli 2En1 harboring recombinant plasmid p2En1, and reference strain E. coli TOP10 harboring the pBK-CMV plasmid

β-Lactam	MIC (μg/ml)
	E. cloacae FFUL2En	E. coli 2En1	E. coli TOP10(pBK-CMV)
Piperacillin + TZBa	>256	>256	3
Cefoxitin	>256	>256	8
Cefuroxime	>256	>256	4
Cefotaxime	>256	8	0.094
Ceftazidime	>256	>256	0.5
Cefepime	0.5	0.38	0.064
Aztreonam	48	6	0.094
Imipenem	0.75	0.38	NDb

^aTZB, tazobactam.

^bND, not determined.

In order to perform the sequencing reactions, the amplicon of 1,234 bp was cloned in the pCR2.1-TOPO vector with a TOPO TA cloning kit, resulting in the plasmid p2En2. The sequence with 382 amino acids has an 86% identity with the AmpC of E. cloacae P99 and 98% identity with the plasmid-borne MIR-1 β-lactamase gene product (2).

To search for a possible chromosomal location of the bla_AmpC gene, whole-cell DNA of E. cloacae FFUL2En was restricted with I-CeuI endonuclease (New England Biolabs), which recognizes a 26-bp sequence in rrn genes coding for the 23S large-subunit rRNA. After digestion, separation of the resulting fragments was performed on a contour-clamped homogeneous electric field-DRII apparatus, as described previously (3).

The restricted fragments of E. cloacae FFUL2En DNA were transferred to a nylon membrane by Southern blotting (9) and were hybridized by using a nonradioactive labeling and detection kit (Roche) with a PCR-obtained probe with primers TN5 and TN2 (see above), consisting of a 576-bp fragment of bla_AmpC and a 16S rRNA gene probe amplified with universal primers described elsewhere (4). The bla_AmpC probe hybridized only with the 630-kb fragment of E. cloacae FFUL2En. These data indicate the chromosomal location of the bla_AmpC gene, coding for the AmpC β-lactamase EcloFFUL2En, in E. cloacae FFUL2En, which is closely related to the plasmid-borne MIR-1 from Klebsiella pneumoniae.